Peptide vaccines against group A streptococci

ABSTRACT

This invention, in one aspect, relates to synthetic immunoreactive peptides. These peptides are approximately 20-25 amino acids in length which are portions of the N termini of the M proteins of the most prevalent United States (U.S.) Group A  Streptococcus  (GAS) serotypes. At least some of the synthetic peptides can be recognized by M type-specific antibodies and are capable of eliciting functional opsonic antibodies and/or anti-attachment antibodies without eliciting tissue cross-reactive antibodies. In another aspect, it relates to compositions or vaccines comprising these synthetic serotype-specific peptides, including polypeptides and proteins. The invention may also be isolated antibodies which are raised in response to the peptides, compositions or vaccines. The invention further relates to kits for using the peptides, compositions, or antibodies. In still further aspects, the invention also relates to methods for using the peptides, compositions, vaccines, or antibodies and methods for tailoring vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 13/427,477,filed Mar. 22, 2012, now U.S. Pat. No. 8,420,107 which is a divisionalof U.S. patent application Ser. No. 12/973,247, filed Dec. 20, 2010, nowabandoned, which is a divisional of U.S. patent application Ser. No.12/144,461, filed Jun. 23, 2008, now U.S. Pat. No. 7,883,710, issuedFeb. 8, 2011, which is a continuation of U.S. patent application Ser.No. 10/477,955, filed Mar. 15, 2004, now U.S. Pat. No. 7,407,664, issuedAug. 5, 2008, which is the §371 U.S. National Stage of InternationalApplication PCT/US2002/015909, filed May 20, 2002, which claims thebenefit of U.S. Provisional Application 60/291,835, filed May 18, 2001,all of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates generally to immunoreactive molecules,compositions, and methods related thereto. Specifically, those relatedto Group A Streptococci. More specifically, it relates to syntheticGroup A strep immunoreactive peptides, compositions comprising thepeptide sequences, vaccines, isolated antibodies elicited by thepeptides, kits comprising the peptides or antibodies, and methods ofusing the peptides, compositions, vaccines and antibodies.

BACKGROUND OF THE INVENTION

Group A streptococci (GAS) are responsible for a wide variety ofdiseases. These range from umcomplicated pharyngitis to more seriousinvasive diseases such as necrotizing fasciitis (“flesh eatingsyndrome”) and streptococcal toxic shock syndrome. Additionally,approximately 3% of GAS infections that go untreated will result inacute rheumatic fever. (Brandt, E. R., Good, M. F. 1999. Vaccinestrategies to prevent rheumatic fever. Immunol. Res. 19:89-103) All agesare susceptible to GAS attack, but those particularly vulnerable are theelderly, children under 2 years, and African Americans. (EmergingInfections Programs (EIP), supported by the National Center forInfectious Diseases for isolates resulting from active surveillance1995-1997. California EIP: Arthur Reingold; Connecticut EIP: MattCarter; Georgia EIP, Monica Farley; Minnesota EIP, Kristine MacDonald;Oregon EIP, Paul Cieslak; Centers for Disease Control and Prevention(CDC), K. O'Brien, B. Beall, K. Deaver-Robinson, R. Facklam, A. Kraus,A. Schuchat, B. Schwartz) Recently, there has been a significantincrease in the number of streptococcal infections (Davies, H. D.,McGeer, A., Schwartz, B., et al. 1996. Invasive group A streptococcalinfections in Ontario, Canada. Ontario group A streptococcal studygroup. N. Engl. J. Med. 335:547-54) as well as rheumatic fever (Veasey,L. G., Wiedneier, S. W., Osmond, G. S., et al. Resurgence of acuterheumatic fever in the intermountain region of the United States. N.Engl. J. Med. 316:42-7). Based on recent active surveillance, it isestimated that there are approximately 8,500 cases and 1,300 deathsannually in the United States from invasive GAS disease, (EIP supportedby the National Center for Infectious Diseases for isolates resultingfrom active surveillance 1995-1997. California EIP: Arthur Reingold;Connecticut EIP: Matt Carter; Georgia EIP, Monica Farley; Minnesota EIP,Kristine MacDonald; Oregon EIP, Paul Cieslak; CDC, K. O'Brien, B. Beall,K. Deaver-Robinson, R. Facklam, A. Kraus, A. Schuchat, B. Schwartz).

A vaccine against GAS could eliminate millions of dollars in health carecosts and numerous physician visits.

There are a number of strategies that have been used towards designingan effective streptococcal vaccine (Salvadori, L. G., Blake, M. S.,McCarty, M., Tai, J. Y., Zabriskie, J. B. 1995. Group Astreptococcus-liposome ELISA antibody titers to group A polysaccharideand opsonophagocytic capabilities of the antibodies. J. Infect. Dis.171:593-600; Ji, Y. Carlson, B., Kondagunta, A., Cleary, P. P. 1997.Intranasal immunization with C5a peptidase prevents nasopharyngealcolonization of mice by group A streptococcus. Infect. Immun.65:2080-2087; Kapur, V. Maffei, J. T., Greer R. S., Li, L. L., Adams, G.J., Musser, J. M. 1994. Vaccination with streptococcal cysteine proteaseprotects mice against challenge with heterologous group A streptococci.Microb. Pathogenesis. 16:443-450; Dale, J. B., Baird, R. W., Courtney,H. S., Hasty, D. L., Bronze, M. S. 1994. Passive protection of miceagainst group A streptococcal pharyngeal infection by lipoteichoic acid.J. Infect. Dis. 169:319-323; Dale, J. B., Washburn, R. G., Marques, M.B., Wessels, M. R. 1996. Hyuaronated capsule and surface M protein inresistance to opsonization of group A streptococci. Infect. Immun.64:1495-1501; Fischetti, V. A. 1989. Streptococcal M protein: moleculardesign and biological behavior. Clin. Microbiol. 2:285-314; Lancefield,R. C. 1962. Current knowledge of the type-specific M antigens of group Astreptococci. J. Immun. 89:307-313; Lancefield, R. C. 1959. Persistenceof type-specific antibodies in man following infection with group Astreptococci. J. Exp. Med. 110:271-283).

There are difficulties associated with a vaccine strategy involving theM protein, such as the large number of serologic M (emm) types (over 100serotypes) and the observation that some M proteins contain epitopesthat cross-react with human tissues. In addition to the large number ofserotypes, every population has a different subset of GAS serotypeswhich are the most prevalent. In order to deal with these difficulties,different approaches have been tried. For example, observation that theM protein's C-terminus is conserved while the N-terminus is variable hasled some workers to try to focus on the C-terminus for broaderprotection and others to focus on the N-terminus where the mostvariability is.

Even though some M protein-based vaccines have been designed, for theabove reasons, a need still exists for a flexible, effective,multivalent GAS vaccine.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates toimmunoreactive peptides. In another aspect, it relates to compositionsor vaccines comprising the peptides, including polypeptides andproteins.

The synthetic peptides of the invention are approximately 20-25 aminoacids in length which are portions of the N termini of the M proteins ofthe most prevalent United States (U.S.) GAS serotypes and which areimmunoreactive. At least some of the synthetic peptides can berecognized by M type-specific antibodies and are capable of elicitingfunctional opsonic antibodies and/or anti-attachment antibodies withouteliciting tissue cross-reactive antibodies.

The invention is also a composition or a vaccine comprised of thesesynthetic serotype-specific peptides of 20-25 amino acids in length fromGAS M proteins. The peptides can be used, for example, individually, ina mixture, or in a polypeptide or protein. Examples of ways thepolypeptide or protein can be created include fusing or linking thepeptides to each other, synthesizing the polypeptide or protein based onthe peptide sequences, and linking or fusing the peptides to a backbone.Also, a liposome may be prepared with the peptides conjugated to it orintegrated within it. The compositions or vaccines may further compriseadditional components, including but not limited to, carriers, vehicles(e.g., encapsulated, liposomes), and other immune-stimulatory molecules(e.g., adjuvants, other vaccines). Additionally, a DNA vaccinecomprising DNA encoding the peptides or compositions of the presentinvention is disclosed.

The invention may also be isolated antibodies which are elicited inresponse to the peptides, compositions or vaccines.

In further aspects, the invention also relates to methods for using thepeptides, compositions, vaccines, or antibodies and methods fortailoring vaccines. The invention still further relates to kits forusing the peptides or antibodies, which can, for example, be used fordiagnostic purposes.

Additional aspects of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. The aspectsof the invention will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing, which is incorporated in and constitute a partof this specification, illustrates several aspects of the invention andtogether with the description, serves to explain the principles of theinvention.

FIG. 1 shows a bar graph of a population-based U.S. sterile siteinvasive isolate distribution among the 24 most prevalent emm types(2321 (88.9%) of the entire 2612 isolate sample) of Group A strep. The10 most prevalent isolates (emm1, emm3, emm28, emm12, emm4, emm11,emm89, st2967, emm77/27L, emm6) account for 65% of the disease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific peptides, specific syntheticmethods, specific compositions, specific vaccines, specific antibodies,specific kits, specific methods of use, as such may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a peptide” includes mixtures of peptides, reference to “acarrier” includes mixtures of two or more carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably and as used herein refer to more than one amino acidjoined by a peptide bond.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

By the term “effective amount” of a compound as provided herein is meanta nontoxic but sufficient amount of the compound to provide the desiredbiological effect. As will be pointed out below, the exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the particular compound used,its mode of administration, and the like. Thus, it is not possible tospecify an exact “effective amount.” However, an appropriate effectiveamount may be determined by one of ordinary skill in the art using onlyroutine experimentation.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the composition in which it is contained.

“Synthetic” is meant to encompass items, e.g., peptides, which are notnaturally occurring, in that they are isolated, synthesized, orotherwise manipulated by man.

“Immunoreactive” as used herein is meant to encompass materials whichare capable of reacting with a specific antigen. “Antigenic” and“immunogenic” are terms which fall within the scope of the term“immunoreactive”.

“Composition” as used throughout the specification and claims is meantto include any composition of matter, including the peptides,polypeptides, proteins, mixtures, vaccines, antibodies or other forms ofmatter of the present invention. It is meant to be used generically andinterchangeably with the other composition of matter terms, and if usedin addition to the other terms, it is used for sake of completeness.“Composition” is a broad term overlapping the coverage of these morespecific terms and when used in addition to these more specific terms itis not meant as an indication that it is necessarily different fromthese more specific terms.

In one aspect, the present invention provides synthetic peptides,compositions, and a vaccine made therefrom and isolated antibodieselicited by administration thereof. The invention also provides methodsfor using the peptides, compositions, vaccines, or antibodies such as,vaccination of recipients. The invention further provides a method fortailoring vaccines. The invention additionally provides kits for usingthe peptides or antibodies.

Peptides

The invention is synthetic peptides of approximately 20-25 amino acidsin length selected from a section of approximately 45 amino acids fromthe most N terminal region of the M proteins of the most prevalent U.S.Group A Streptococcus (GAS) serotypes which are immunoreactive. At leastsome of the peptides are capable of eliciting opsonic antibodies and/oranti-attachment antibodies to the GAS serotypes without eliciting tissuecross-reactive antibodies. In one aspect of the invention, the syntheticpeptides are from the most prevalent invasive U.S. GAS serotypes whichare immunoreactive. The prevalence data in FIG. 1 includes data frominvasive isolates. The most frequently occurring invasive types reflectthe incidence rate of the same types found in non-invasive isolates.Specific peptides of the present invention are shown below in Table 1.One aspect of the invention is a peptide consisting essentially of theamino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59,SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79,SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99,SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113,SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ IDNO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127,SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ IDNO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQID NO:137, or SEQ ID NO:138.

Examples of the peptides of the invention, several for each of the 25most common serotypes (138 peptides), are as follows:

TABLE 1 Synthesized Type-Specific Peptides. Seq Peptide ID design- No:Serotype ation Peptide 1 M1 M1-1 CNGDGNPREVIEDLAANNPAIQ 2 M1-2CIQNIRLRHENKDLKARLENA 3 M1-3 CIRLRHENKDLKARLENAMEV 4 M1-4CNGDGNPREVIEDLAANNPAME 5 M1-5 CIRNIRLRHENKDLKARLENA 6 M1-6CNGDGNPREVIEDLAANNPVIQ 7 M1-7 CNGDGNPRVVIEDLAANNPAIQ 8 M1-8CIRLRHHENKDLKARLENAMEV 9 M2 M2-1 CNSKNPVPVKKEAKLSEAELHDK 10 M2-2CKKEAKLSEAELHDKIKNLEEEK 11 M2-3 CELHDKIKNLEEEKAELFEKLD 12 M2-4CELFEKLDKVEEEHKKVEEEHKK 13 M3 M3-1 CDARSVNGEFPRHVKLKNEIE 14 M3-2CGEFPRHVKLKNEIENLLDQV 15 M3-3 CLDQVTQLYTKHNSNYQQYNA 16 M3-4CLDQVTQLYNKHNSNYQQYSA 17 M3-5 CLDQVTQLYTKHNSNYQQYSA 18 M3-6CLNQVTQLYTKHNSNYQQYNA 19 M3-7 CLAQVTQLYTKHNSNYQQYNA 20 M3-8CLNQVTQLHTKHNSNYQQYNA 21 M3-9 CRSDARSVNGEFPRHVKLKNE 22 M3-10CQLYTKHIYTKHNSNYQQYNAQ 23 M3-11 CTQLYTKHNSNYQQYNAQAGR 24 M4 M4-1CAEIKKPQADSAWNWPKEYNA 25 M4-2 CDSAWNWPKEYNALLKENEEL 26 M4-3CKENEELKVEREKYLSYADDK 27 M4-4 CEELKVEREKYLSYADDKEKDPQ 28 M11 M11-1CAGQSAPKGTNVSADLYNSLWDE 29 M11-2 CKGTNVSADLYNSLWDENKT 30 M11-3CDENKTLREKQEEYITKIQNE 31 M11-4 CTEVKAAGQSAPKGTNVSADL 32 M12 M12-1CDHSDLVAEKQRLEDLGQKFE 33 M12-2 CAEKQRLEDLGQKFERLKQRS 34 M12-3CLEDLGQKFERLKQRSELYLQ 35 M12-4 CKFERLKQRSELYLHQYYDNK 36 M12-5CKFERLKRRSELYLQQYYDNK 37 M12-6 CKQRSELYLQQYYDNKSNRYK 38 M12-7CSELYLQQYYDNKSNGYKGDW 39 M22 M22-1 CESSNNAESSNISQESKLINT 40 M22-2CESSNISQESKLINTLTDENEK 41 M22-3 CESKLINTLTDENEKLREELQQ 42 M22-4CNTLNTLTDENEKLREELQQ 43 M22-5 CESSNISQESKLINTLTDENEK 44 M22-6CEKLREELQQYYALSDAKEEE 45 M28 M28-1 CAESPKSTETSANGADKLAD 46 M28-2CKSTETSANGADKLADAYNTL 47 M28-3 CDKLADAYNTLLTEHEKLRDE 48 M28-4CTEHEKLRDEYYTLIDAKLEEK 49 M28-5 CTEHEKLRDEYYTLIDAKEEE 50 M77 M77-1CEGVSVGSDASLHNRITDLEEEREK 51 M77-2 CSDASLHNRITDLEEEREKLLNK 52 M77-3CDLEEEREKLLNKLDKVEEEHKKD 53 M77-4 CDLEEERGKLLNKLDKVEEEHK 54 M77-5CLNKLDKVEEEHKKDHEQLEK 55 M89 M89-1 CDSDNINRSVSVKDNEKELHNK 56 M89-2CDNINRSVSVKDNEKELHNKIAD 57 M89-3 CSVKDNEKELHNKIADLEEER 58 M89-4CELHNEIADLEEERGEHLDKID 59 M89-5 CELHNKIADLEEERGAHLDKID 60 M89-6CDSDNINRFVSVKDNEKELHN 61 M89-7 CDSDNSDNINRSVSVKDNEKE 62 M89-8CLEEERGEHLDKIDELKEELK 63 st2967 st2967-1 CNSKNPAPAPASAVPVKKEATK 64st2967-2 CVPVKKEATKLSEAELYNKIQ 65 st2967-3 CKKEATKLSEAELYNKIQELEE 66st2967-4 CNSKNPAPAPAVPVKKEATKL 67 st2967-5 CNSKNPAPAVPVKKEATKLSE 68st2967-6 CAELYNKIQELEEGKAELFDK 69 M6 M6-1 CRVFPRGTVENPDKARELLNK 70 M6-2CRGTVENPDKARELLNKYDVEN 71 M6-3 CENPDKARELLNKYDVENSMLQ 72 M6-4CENSMLQANNDNLTDQNKNLTD 73 M6-5 CNSMLQANNDKLTTENKNLTD 74 M82 M82-1CDSSSRDITEAGVSKFWKSKFD 75 M82-2 CRDITEAGVSKFWKSKFDAEQN 76 M82-3CEAGVSKFWKSKFDAEQNRANE 77 M82-4 CDAEQNRANELEKKLSGYEKD 78 M43 M43-1CEEHPDVVAARESVLNNVR 79 M43-2 CHPDVVAARESVLNNVRVPGT 80 M43-3CRVPGTLWLRQKEENDKLKLEK 81 M43-4 CLRQKEENDKLKLEKKGLETE 82 M75 M75-1CEEERTFTELPYEARYKAWKSE 83 M75-2 CELPYEARYKAWKSENDELREN 84 M75-3CNDELRENYRRTLDKFNTEQ 85 M75-4 CKAWKSENDELRENYRKTLDK 86 M75-5CRENYRRTLDKFNTEQGKTTR 87 M33 M33-1 CEEHEKVTQAREAVIREMQQR 88 M33-2CHEKVTQAREAVIREMQQRGT 89 M33-3 CEMQQRGTNFGPLLASTMRDNH 90 M92 M92-1CDDRSVSTNSGSVSTPYNNLLNE 91 M92-2 CRSVSTNSGSVSTPYNNLLNE 92 M92-3CEYDDLLAKHGELLSEYDALK 93 M92-4 CDLLAKHGELLSEYDALKEKQDK 94 M5 M5-1CTVTRGTINDPQRAKEALDKYE 95 M5-2 CDPQRAKEALDKYELENHDLK 96 M5-3CENHDLKTKNEGLKTENEGLK 97 M5-4 CQRAKAALDKYELENHDLKTKN 98 M5-5CTVTRGTVNDPQRAKEALDKYE 99 M5-6 CTVTRGTVNDPQRAKETLDKYE 100 M5-7CTVTRGTINDPQRAKEVIDKYE 101 M5-8 CTVTRSTINDPQRAKEALDKYE 102 M5-9CHDLKTKNEGLKTENEGLKTEN 103 M94 M94-1 CEEASNNGQLTLQHKNNALTSE 104(formerly M94-2 CQHKNNALTSENESLRREKDR 105 emm13W) M94-3CESLRREKDRYLYEKEELEKK 106 M94-4 CRREEKDRYLYEKEELEKKNK 107 M73 M73-1CDNQSPAPVKKEAKKLNEAE 108 M73-2 CKKEAKKLNEAELYNKIQELE 109 M73-3CELYNKIQELEEGKAELFDKLEK 110 M73-4 CDNQSPALVKKEAKKLNEAEL 111 M73-5CDNQSPAPAPVKKEAKKLNEAE 112 M73-6 CQELEEGKAELFDKLEKVEEE 113 M18 M18-1CAAPLTRATADNKDELIKRAND 114 M18-2 CRATADNKDELIKRANDYEIQ 115 M18-3CEIQNHQLTVENKKLKTDKEQ 116 M18-4 CRATADNKDELIKRANGYEIQ 117 M18-5CKDELIKRKELTIIEIQNHQL 118 M18-6 CNHQLTVENKKLKTDKEQLTKE 119 M58 M58-1CDSSREVTNELTASMWKAQAD 120 M58-2 CREVTNELTASMWKAQADSAK 121 M58-3CKAKELEKQVEEYKKNYETLEK 122 M58-4 CDSSREVTNELAASMWKAQAD 123 M58-5CDSSRDSSREVTNELTASMWK 124 M58-6 CKAKELEKQVEEYKKNYETLEK 125 M59 M59-1CEQAKNNNGELTLQQKYDALT 126 M59-2 CELTLQQKYDALTNENKSLRRE 127 M59-3CNENKSLRRERDNYLNYLYEK 128 M59-4 CRRERDNYLNYLYEKEELEKK 129 M101 M101-1CADHPSYTAAKDEVLSKFSVPGH 130 (formerly M101-2 CKDEVLSKFSVPGHVWAHERE 131stNS5) M101-3 CHEREKNDKLSSENEGLK 132 M101-4 CDKLRLEKEELKTDLQKKERE 133M101-5 CKNDKLSSENEGLKAGLQEKE 134 M41 M41-1 CEGNARLAQAQEEALRDVLNN 135M41-2 CRLAQAQEEALRDVLNNTPHN 136 M41-3 CQAQEEALRDVLNNTPHNQLRD 137 M41-4CDVLNNTPHNQLRDAYAGAFRR 138 M41-5 CQLRDPYAGAFRRNNELEKIIQE

It is important to note that a single peptide representing each of the25 M serotypes represented is predicted to protect against the majorityof invasive GAS within each of these serotypes in the U.S. For themajority of these types, the invention provides a peptide that actuallymatches the sequences of all GAS of these types that we haveencountered. We include additional peptides that encompass the extent ofM protein gene allelic variation that we have encountered to date withineach type from various geographic locations. It is important to notethat for the majority of these types, at least one peptide is conservedamong all allelic variants that we have encountered.

The small size of the peptides used in the current invention allows aflexible approach for formulating compositions or vaccines. Theformulations can be readily and inexpensively changed to account forchanges in GAS serotype frequencies in the target population. Theadaptability includes frequency changes between populations, years, andthe like. Any population for which there is frequency data can have avaccine formulation customized for it by the methods of the presentinvention, which are discussed below.

Recently a rapid M protein gene-based subtyping system has beeninitiated which predicts the type-specific portion of the M protein withvery high efficiency. (Beall, B., Facklam, R., Hoenes, T., Schwartz, B.1997. A survey of emm gene sequences from systemic Streptococcuspyogenes infection isolates collected in San Francisco, Calif.; Atlanta,Ga.; and Connecticut state in 1994 and 1995. J. Clin. Microbiol.35:1231-1235; Beall, B., Facklam, R., Elliot, J., Franklin, A., Hoenes,T., Jackson, D., Laclaire, L., Thompson, T., Viswanathan, R. 1998.Streptococcal emm types associated with T-agglutination types and theuse of conserved emm restriction fragment patterns for subtyping group AStreptococci. J. Med. Micro. 47:1-6). A Centers for Disease Control andPrevention (CDC) surveillance system used this rapid gene based Msubtyping system to gather epidemiological data which showed that the 30most prevalent invasive serotypes account for approximately 95% of thetotal invasive isolates in the U.S.

In addition, these peptides should have direct use in formulatingvaccines for countries other than the U.S. For example, the 25 serotypesrepresented in Table 1 also appear to encompass the majority of GASpediatric pharyngitis isolates in Rome, Italy (91/114=80%), 85%(367/430) of a mixture of sterile and non-sterile GAS isolates recoveredin Mexican patients, and 80% (110/137) of primarily invasive isolatesrecently recovered from patients in Argentina.

It is also important to note that data indicates that these 25 serotypeswould have less coverage in other geographic areas such as Malaysia,India, New Guinea, Nepal, and Egypt. For example, out of 136 pharyngitisand impetigo isolates recently recovered in Egypt, only 62 (46%) were ofone of these 25 types. While type emm1 is by far the most prevalent typerecovered from invasive and noninvasive U.S. isolates (about 20%), only5/136 (4%) Egypt isolates were type emm1. Thus, the methods of thepresent invention could be used to tailor vaccines or compositions withthe serotypes most prevalent in these areas.

The peptides are synthesized by any of the techniques known in the art,as the method of making them is not critical. One technique is throughrecombinant methods. Another is manual or automated chemical synthesisusing individual amino acids, such as solid phase peptide synthesis.Other methods for synthesizing peptides may be readily apparent to oneof ordinary skill in the art.

One of ordinary skill in the art would be able to determine throughroutine experimentation which of the immunoreactive peptides are capableof eliciting opsonic and/or anti-attachment antibodies.

Though it is known generally in the art that even single substitutionsmay have a great impact on immunogenicity of a molecule, due to allelicvariants which exist for any particular GAS serotype, there are expectedto be allowable substitutions within the peptides corresponding witheach serotype which maintain immunogenicity. As discussed above, theexample peptides in Table 1 include allelic variants of the peptides fora given serotype. For example, up to approximately 3 substitutionswithin each peptide which correspond with variants of a given serotypemay create peptides which also are immunoreactive. That a givensubstitution results in an immunoreactive peptide can be determined byroutine experimentation by making a proposed substitution then testingthe immunoreactivity by one of many known assays including thosedescribed herein. A variant within a serotype can be identified on thebasis of sequence. Any variation within 50 N-terminal residues of matureprotein of M protein gene type strain is considered a variant. Isolateswithin an emm type share about ≧84% deduced amino acid sequence identity[as determined by the Wisconsin Package Version 10.1, Genetics ComputerGroup (GCG), Madison Wis. FASTA program] within the mature aminoterminal 45 amino acids compared to the reference type strain sequence.The first three peptides indicated for each serotype in Table 1 areconsidered to be the peptides from the majority of isolates of theserotype. The additional peptides given in Table 1 for each serotype insome instances represent the majority of isolates in the type, and inother instances represent known variants of these types.

At least some of the individual peptides are capable of protecting arecipient against its corresponding serotype. A composition comprising amixture of peptides from more than one serotype is able to protectagainst those corresponding serotypes. A mixture can be tailored suchthat it contains the most prevalent serotypes in an area (population),thus making the mixture able to protect against the most importantserotypes. The tailoring is accomplished by matching theserotype-specific peptides to epidemiological data regarding theprevalence of the serotypes for the population of recipients desired tobe protected.

Though each peptide will be immunoreactive for the serotype upon whichit is based, the peptides of the present invention may even providenon-serotype-specific effects. It is believed that it is possible thatcertain prevalent N-terminal fragments may evoke cross-protectiveopsonic antibodies. This is demonstrated in Example 5 below. It isexpected that the present peptides, compositions or vaccines will evokecross-type opsonization.

Compositions, Vaccines, and Kits

The invention is also polypeptides, proteins, compositions, or vaccinescomprising the peptides or sequences of the peptides. The peptides, inaddition to being used individually, can be used as a mixture ofpeptides. One aspect of the invention is a composition comprising thepeptides of the present invention as described above. A compositioncomprising a mixture of peptides is readily prepared by methods wellknown in the art. Alternatively, to using the peptides individually orin a mixture, the peptides may be joined together into a polypeptide orprotein. One aspect of the invention is a polypeptide comprising thesequences of peptides of the present invention. Another aspect of theinvention is a protein comprising the sequences of the peptides of thepresent invention. Standard techniques known in the art may be used to,for example, link the synthesized peptides, synthesize a polypeptide orprotein which contains segments corresponding to the desired syntheticpeptides, or link the synthetic peptides to a backbone or a liposome.Examples of backbones include, for example, keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, diphtheria toxoid, bacterial outermembrane proteins, and artificial amino acid backbones. It is well knownto one of ordinary skill in the art how to covalently bond peptides to abackbone or liposome or how to create polypeptides or proteins usingrecombinant techniques.

As noted above, a vaccine comprising these synthetic peptides is withinthe scope of the invention. In one aspect, the vaccine comprises animmunogenic amount of the peptide immunogens of the present invention.The data from a CDC surveillance system showing the epidemiologicaldata, as noted above, showed that the 30 most prevalent invasive M typesaccount for approximately 95% of the total invasive isolates in the U.S.An aspect of the present invention is the development of amulti-antigenic peptide (MAP) vaccine representing these most prevalentserotypes. The peptides of the invention may be conveniently formulatedinto vaccine compositions comprising one or more of the peptides aloneor in association with a pharmaceutically acceptable carrier. See, e.g.,Remington's Pharmaceutical Sciences, latest edition, by E. W. MartinMack Pub. Co., Easton, Pa., which discloses typical carriers andconventional methods of preparing pharmaceutical compositions that maybe used in conjunction with the preparation of formulations of theinventive peptides and which is incorporated by reference herein. Abenefit of the vaccine is it can eliminate over 85% of Group AStreptococci infections and reduce by 85% the nasopharyngeal reservoirof Group A Streptococci in the United States with the correct tailoring.The reservoir of GAS is expected to be reduced for the population, notjust an individual. Reduction in GAS would have an effect on carriage ofthe organism, thereby affecting the reservoir in the population.Reduction in carriage of the organism subsequently reduces the exposurerate, thereby increasing herd immunity.

The vaccine comprises and can be made by providing immunogenic amountsof the peptides alone or in a pharmaceutically acceptable vehicle orcarrier. Carriers include water, saline, dextrose, and glycerol, forexample. The vaccine can further comprise additional immune-stimulatorymolecules, including other GAS immunogens, vaccines of other species(such as H. influenza, pertussis, N. meningitidis, pneumococcus, orInfluenzae), and adjuvants or mixture of adjuvants. One of ordinaryskill in the art would be able to identify vehicles, carriers, otherantigens or immunogens, and immunomodulators, such as adjuvants orcytokines, appropriate for the present invention. Additional additiveswould also be readily apparent to one of skill in the art, such aswetting agents or preservatives.

A DNA vaccine is also within the scope of the present invention. Oneaspect of the invention is a DNA vaccine comprising DNA encodingimmunoreactive peptides or compositions of the present invention.Methods for making DNA sequences suitable for DNA vaccines are known inthe art. One of ordinary skill would be able to determine appropriatepromoters or other regulatory sequences which may be used in the DNAconstruct encoding the immunoreactive compositions. DNA vaccines mayfurther comprise other components as in the vaccines and compositionsdescribed above and below, such as carriers and agents which increaselevels of immunity, such as liposomes. DNA vaccines may be administeredby routes similar to other vaccines. Administration of a DNA vaccineresults in expression of antigens which produce a protective immuneresponse.

Though the vaccine of the present invention is expected be mosteffective with multiple serotype-specific peptides, it could containfrom one serotype-specific peptide to multiple serotype-specificpeptides for every identified serotype of GAS. One of skill in the artwould be able to determine the most cost-effective and clinicallytherapeutic combination based on epidemiological data, using thetailoring method provided herein. In one aspect of the invention, thevaccine contains at least 3 serotype-specific peptides from 3 differentserotypes. For example, a vaccine comprising serotype-specific peptidesfor emm1, emm3, and emm12 is expected to protect against approximately38% of invasive GAS disease in the U.S. More specifically, this vaccinecan comprise the following peptide combinations from Table 1:

M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-1M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-1 M12-1 M12-1M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-1 M12-1 M12-1 M12-1 M12-1M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-1M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-1 M12-1 M12-1M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-1 M12-1 M12-1 M12-1 M12-1M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-2 M12-2 M12-2M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-2 M12-2 M12-2 M12-2 M12-2M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-2M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-2 M12-2 M12-2M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-2 M12-2 M12-2 M12-2 M12-2M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-3 M12-3 M12-3M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-3 M12-3 M12-3 M12-3 M12-3M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-3M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-3 M12-3 M12-3M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-3 M12-3 M12-3 M12-3 M12-3M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-4 M12-4 M12-4M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-4 M12-4 M12-4 M12-4 M12-4M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-4M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-4 M12-4 M12-4M12-4 M12-1 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-4 M12-4 M12-4 M12-4 M12-4M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-5 M12-5 M12-5M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-5 M12-5 M12-5 M12-5 M12-5M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-5M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-5 M12-5 M12-5M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-5 M12-5 M12-5 M12-5 M12-5M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-6 M12-6 M12-6M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-6 M12-6 M12-6 M12-6 M12-6M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-6M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-6 M12-6 M12-6M12-6 M12-1 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-6 M12-6 M12-6 M12-6 M12-6M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M3-1 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-7 M12-7 M12-7M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-7 M12-7 M12-7 M12-7 M12-7M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5M3-5 M3-5 M3-5 M3-5 M3-5 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6M3-6 M3-6 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-7M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-7 M12-7 M12-7M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-7 M12-7 M12-7 M12-7 M12-7M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-11 M3-11 M3-11M3-11 M3-11 M3-11 M3-11 M3-11 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7M12-7In another aspect of the invention, the vaccine comprises about 10serotype-specific peptides, each peptide corresponding to one of the 10most prevalent serotypes in the U.S., thus making it expected toimmunize against approximately 65% of GAS disease in the U.S. Morespecifically, this vaccine can comprise combinations of 10 peptideswherein one peptide comes from each of the M1, M3, M28, M12, M4, M11,M89, st2967, M77/27L, M6 peptides from Table 1. As demonstrated above,the combinations can be generated and tested according the proceduresdescribed in this application to determine those which are effective. Ina further aspect of the invention, the vaccine comprises about 30serotype-specific peptides of the 30 most prevalent serotypes, thusmaking it expected to immunize against approximately 95% of GAS diseasein the U.S. More specifically, this vaccine can comprise combinations of30 peptides wherein one peptide comes from each of the 30 most prevalentserotypes. As demonstrated above, the combinations can be generated andtested according the procedures described in this application todetermine those which are effective. In a still further aspect of theinvention, the vaccine can comprise at least one serotype-specificpeptide from any identified serotype of GAS. A vaccine coveringapproximately 60% of GAS disease would be expected to be commerciallyviable. FIG. 1 shows the most prevalent serotypes in the U.S. currentlyfrom which the serotype-specific peptides could be chosen to target.Similar data from any targeted population could be used to tailor thevaccine for the prevalent serotypes and a given percentage of disease.This strategy towards a safe and effective vaccine against GAS offersthe advantage of being easily modified to fit the needs of a particularregion according to the predominant M types located there.

As indicated above, based on the current epidemiological data, similarserotype-specific peptides would be expected to be effective in vaccinesor compositions in the U.S., Italy, Mexico and Argentina, for example.The epidemiological data of Malaysia, India, New Guinea, Nepal and Egyptindicate that vaccines or compositions tailored to these areas mayrequire a different subset of GAS serotype-specific peptides. Based onthe teaching herein, such a vaccine is easily within the grasp of theskilled person.

Another strategy for designing a vaccine would be to make it selectivefor specific GAS illnesses, as all GAS do not cause the same illnesses.For example, the most severe GAS diseases are often considered to benecrotizing fasciitis and toxic shock syndrome which are most frequentlycaused by M1 and M3. Thus, selecting immunogenic molecules specific tothese serotypes would tailor the vaccine to this strategy. Morespecifically, the combinations could be, for example,

M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1M3-1 M3-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2M3-2 M3-2 M3-2 M3-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-3 M3-3M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6M1-7 M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M1-1 M1-2 M1-3 M1-4M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M1-1 M1-2M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8M3-8 M3-8 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-9 M3-9 M3-9 M3-9M3-9 M3-9 M3-9 M3-9 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10 M3-10M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11

The peptides, compositions, vaccines or antibodies (discussed below) ofthe present invention may be administered by any mode of administrationcapable of delivering a desired dosage to a desired location for adesired biological effect which are known to those of ordinary skill inthe art. One of ordinary skill would be able to determine these dosagesand routes by routine experimentation. Routes or modes include, forexample, orally, parenterally (e.g., intravenously, by intramuscularinjection, by intraperitoneal injection), or the like, althoughsubcutaneous administration is preferred. Though the vaccine isenvisioned as an injectable, such as subcutaneous or intramuscularly,the vaccine may be formulated in such a way as to render it mucosallydeliverable without the peptides being broken down before providingsystemic or mucosal immunity, such as, orally, inhalationally,intranasally, or rectally. The amount of active compound administeredwill, of course, be dependent, for example, on the subject beingtreated, the subject's weight, the manner of administration and thejudgment of the prescribing physician. Immunogenic amounts can bedetermined by standard procedures. Examples of other peptide vaccinesare known in the art. Dosages of the present invention are expected tobe in similar ranges.

Depending on the intended mode of administration, the compositions orvaccines may be in the form of solid, semi-solid or liquid dosage forms,such as, for example, tablets, suppositories, pills, capsules, powders,liquids, suspensions, or the like, preferably in unit dosage formsuitable for single administration of a precise dosage. The compositionsor vaccines may include, as noted above, an effective amount of theselected immunogens in combination with a pharmaceutically acceptablecarrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc.

A more recently revised approach for parental administration involvesuse of a slow release or sustained release system, such that a constantlevel of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, whichis incorporated by reference herein. A system using slow release orsustained release may be used with oral administration as well. Thevaccine or composition may be administered in liposomes, encapsulated,or otherwise protected or formulated for slower or sustained release.

A subject can be inoculated to generate an active immune response to thepresence of the immunogenic composition which can later protect thesubject from the organism. A passive immune response may be accomplishedby any method known in the art.

Kits using peptides or antibodies produced by the present invention maybe made. A kit comprises packaging and the antibodies or peptides. A kitmay further comprise a solid phase or substrate to which the antibodiesor peptides may be attached.

Antibodies

Antibodies are also within the scope of the invention. For example,isolated antibodies which selectively bind with the peptides of thepresent invention are an aspect of the present invention. Theseantibodies can be used, for example, in diagnosis, treatment, orvaccination techniques. The antibodies can be monoclonal or specificantibodies. The antibodies can be opsonic antibodies or anti-attachmentantibodies. The antibodies are made and isolated by methods well knownin the art. Modified antibodies, fragments and humanized antibodies arealso within the scope of this invention. It is well known in the art howto make and use modified antibodies, fragments or humanized antibodies.

Methods and Uses

The peptides, compositions, vaccines, and antibodies of the presentinvention may be used in a variety of applications. For example,preventative/prophylactic, therapeutic, or diagnostic methods; affinitychromatography for separating/purifying antibodies or antigens;active/passive immunotherapy; and use of antibodies generated in passiveimmunotherapy.

An example of a method of preventing GAS infection comprisesadministering a prophylactically effective amount of vaccine, or of ananti-idotype antibody to the peptides of the present invention, to asubject. Also, the antibodies against the peptides of the presentinvention may be administered in a prophylactically effective amount.

An example of a method of treating a GAS infection comprisesadministering a therapeutically effective amount of antibodies of thepresent invention to a subject.

An example of a diagnostic method is determining the serotype of GASorganism responsible for an infection by contacting a sample withmultiple serotype-specific antibodies of the present invention anddetermining which of these serotype-specific antibodies are actuallybound with the infecting organism. An example of another diagnosticmethod is contacting a sample with multiple serotype-specific peptidesof the present invention and determining which serotype-specificpeptides are actually bound with antibodies in the sample.

A method of measuring the amount of GAS organism in a sample comprisingcontacting a sample with antibodies of the present invention andmeasuring the amount of immunocomplexes formed.

Affinity chromatography is frequently used for separating and/orpurifying antibodies or antigens. By binding the corresponding antibodyor antigen to a substrate, a sample can be passed through a columncontaining the immunoadsorbent and then the column eluted to collect theisolated corresponding antigen or antibody. More specifically, thepeptides of the invention can be bound on a column to purify anti-GASantibodies. Likewise, anti-GAS antibodies generated in accordance withthe invention can be bound to a column and used to purify GAS from asample.

Immunotherapy is another use for the peptides, compositions, vaccines orantibodies of the present invention. As known in the art, activeimmunotherapy is achieved by activating a subject's own immune system.By administering the peptides, compositions or vaccines of the presentinvention, an active immune response may be elicited.

As known in the art, passive immunotherapy is achieved by supplementinga subject's immune system with agents such as antibodies. Byadministering the antibodies of the present invention, a passive immuneresponse may be elicited.

The method for tailoring vaccines comprises a) identifying a populationof recipients for the vaccine; b) gathering prevalence data on serotypesof the targeted organism from a sample within that population ofrecipients; c) choosing a set of the most prevalent serotypes from thegathered data; d) identifying proteins from the chosen serotypesresponsible for evading opsonophagocytosis; e) identifying smallpeptides within the identified proteins which protect for the chosenserotypes; f) synthesizing the identified peptides; g) formulating avaccine comprising the peptides identified in step e). Specifically, thesmall peptides may be those of about 20-25 amino acids and protectionmay be by elicitation of opsonic or anti-attachment antibodies.

Other uses for or variations of the above methods using the abovepeptides, compositions, vaccines or antibodies may be readily apparentto one of ordinary skill in the art.

The approach of employing a mixture of defined synthetic N terminal Mprotein segments protecting against prevalent U.S. Group A streptococcal(GAS) strains will favorably compare against any of the prior artapproaches. The present approach has found excellent immunogenicity andtype-specific opsonic antibody titers with the peptides assessed. Animalstudies have indicated that individual peptides protect in atype-specific manner not only against systemic infection, but againstnasopharyngeal carriage of GAS. Many N terminal M protein segments havealready been demonstrated to not evoke antibodies cross-reactive withhuman tissues. There is no evidence that chemically linking the currentpeptides to carriers or backbones will increase the risk of undesirablecross reactions. The methodology can be proven for each of the mostcommon M types found in U.S. invasive disease isolates. The strategy canbe expanded to less frequently occurring GAS types. This allows thevaccine to be quickly and precisely adapted to changes in individualstrain frequencies in a given geographic area or demographic populationby addition or deletion of individual peptide components.

EXAMPLES Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods claimedherein are made and evaluated, and are intended to be purely exemplaryof the invention and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Materials and Methods

A CDC surveillance system used a rapid gene-based M subtyping system togather epidemiological data and showed that the 30 most prevalentinvasive M types account for approximately 95% of the total invasiveisolates in the U.S.

Peptide Synthesis and Purification

Synthetic peptides (approximately 20-25-mers), representing epitopes ofthe most prevalent GAS M types were synthesized. Their sequences werederived from the M protein N-terminal region that confers type-specificimmunity.

Peptides were synthesized on a Model ACT396 Omega multiple peptidesynthesizer (Advanced Chemtech, Louisville, Ky.) by Fmoc-chemistry usinga HOBT/DIC strategy with double couplings. Matrix-assisted laserdesorption/ionization, time-of-flight mass spectroscopy (MALDI-TOF MS)(Bruker REFLEX, Billerica, Mass.) was utilized to determine the mass tocharge (m/z) of the crude peptides.

Abbreviations:

Fmoc (9-fluorenylmethyloxycarbonyl)

HOBT (1-hydroxybenzotriazole)

DIC (Diisopropylcarbodiimide)

Enzyme-Linked Immunosorbent Assay (ELISA)

Overlapping peptides representing each M type were assessed for theirimmunogenicity using ELISA and dot-immunoblot.

Levels of anti-M protein antibodies in mouse sera were determined by anenzyme-linked immunosorbent assay (ELISA) using either syntheticpeptides or whole M protein as antigen. Microtiter plates (DYNEX Immulon2 HB) were coated with 10 μg/ml antigen in 10 mM phosphate-bufferedsaline (PBS) at 4° C. overnight. Plates were rinsed three times withwash buffer (0.05% Tween 20 in 10 mM PBS, pH 7.2) and blocked with 1%BSA in PBS for one hour at 37° C. The appropriate test sera were appliedat a starting dilution of 1:1000 and serially diluted two fold down theplate in PBS. Plates were incubated at 37° C. for 30 minutes andsubsequently rinsed three times. Goat anti-rabbit or goat anti-mouseIg-peroxidase conjugate diluted 1:10,000 in PBS was added to all wellsand incubated for two hours at 37° C. Plates were rinsed three timeswith wash buffer and freshly prepared TMB peroxidase (Kirkegaard andPerry Laboratories) was added. Reactions were quenched with 0.18 M H₂SO₄after 30 minutes at room temperature and OD₄₅₀ was measured with aLabsystems Multiskan plate reader using Ascent software. ELISA titersare expressed as the reciprocal of the last dilution which gave areading OD₄₅₀>0.10.

Immunization

Mice (6 week old female Swiss Webster, Harley-Sprague) were immunizedsubcutaneously (s.c.) with either the peptide in alum or alum alone forcontrol groups. Peptides were rotated in the alum mixture for 2 hours at4° C. and stored at 4° C. overnight prior to use. Initial injections(100 μl) consisted of 50 μg peptide in alum (25 μg for M3-2 peptide)with identical booster injections given at 2 and 4 weeks (except for theM3-2 peptide which was only given as an initial dose and single boosterat 2 weeks).

Opsonophagocytosis

Opsonophagocytosis assays were performed as previously described inLancefield, R. C., Persistence of type-specific antibodies in manfollowing infection with group A streptococci, 1959, J. Immunol.,89:307. Briefly, diluted serum (50 μl) was added to mid-log phase GAS(10³ CFU) in Todd-Hewitt broth (50 μl) and whole, heparinized blood (500μl) from a nonopsonic human donor. Mixtures were briefly vortexed andplaced at 37° C. in a shaker for 3 hours. Dilutions were then plated ontrypticase soy agar plates (5% sheep blood) to quantitate viableorganisms. The % killing is expressed as [(CFU control-CFU test)/CFUcontrol]×100.

NP Challenge

Mice were challenged intranasally one week after the final boosterinjection was administered. Prior to being challenged, mice wereanesthetized with a ketamine/xylazine mixture. Mice were then given10⁴-10⁵ CFU (10 μl) of streptococci intranasally via a microliterpipette. They were sacrificed 24 hours after the challenge and thenasopharyngeal passages were washed with approximately 100 μlphysiological saline which was collected and immediately placed on ice.Dilutions of the wash were then plated on trypticase soy agar plates (5%sheep blood) and incubated at 37° C. for 18 hours to quantitate viableorganisms. Nasopharyngeal colonization in immunized mice was compared tonon-immunized controls. Statistical analysis of the NP data wasaccomplished using the t-test and rank sum test.

Detection of Heart Cross-Reactive Antibodies

Mouse sera were screened for heart cross-reactive antibodies with anindirect immunofluorescence assay (IFA). Glass slides containingformaline-fixed human heart tissue were deparaffinized before use andstored in dH₂O. Slides were air-dried 10 minutes and incubated in amoist chamber with 1:500 mouse test sera at room temperature for 30minutes. Slides were rinsed and soaked in PBS for 5 minutes. Slides werethen incubated in a moist chamber with 1:500 goat anti-mouseFITC-labeled globulin at room temperature for 30 minutes. Slides werewashed as described previously and allowed to air-dry. One drop ofmounting fluid (DAKO) was applied to each slide followed by a glasscover slip (Corning 24×40 mm). Slides were immediately examined under afluorescent microscope. The positive control was a 1:500 mouseanticlonal antibody to human HLA (Caltag).

Example 1 Peptide Recognition by Anti M-Protein Rabbit Sera

The immunoreactivity of several synthetic peptides was determined byELISA using rabbit sera prepared against whole M protein. The syntheticpeptides are representative of relatively small portions of the Mprotein N-terminus; therefore, it was advantageous to determine if theepitopes contained within particular peptides were immunoreactive withsera prepared against whole M protein. It was shown that the wholeanti-M protein rabbit sera of the respective serotypes could bind toeach of the synthetic peptides within that serotype. However, somepeptides were more highly immunoreactive than others, thus being bettersuited for use in the animal studies. The peptides showing the highestreactivity in each serotype group tested were M1-4, M3-2 and M12-1. Thisindicated that immunoreactive epitopes were contained within the aminoacid sequences of the synthetic peptides.

The immunoreactivity of peptides with SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115,SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ IDNO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ IDNO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 areconfirmed by ELISA using rabbit sera prepared against whole M protein.

Example 2 Immunogenicity of Synthetic Peptides in Swiss Webster Mice

Studies were then carried out in vivo in a mouse model to determine theability of the tested synthetic peptides to protect against GASchallenge and to evaluate their immunogenicity as a single MAPformulation.

Mice were immunized s.c. with selected peptides using aluminum hydroxideor aluminum phosphate as an adjuvant. Serology results indicated thataluminum hydroxide elicited higher antibody levels and, therefore, itwas used for all subsequent immunizations. ELISA indicated thatantibodies were elicited to the M type peptides.

TABLE 2 ELISA results determining elicitation of antibodies. PeptideELISA M1-4 no M1-2 no M1-3 yes M3-1 yes M3-2 yes M3-4 no M12-1 yes M12-2yes (weak) M12-3 no

Five of the nine synthetic peptides shown in Table 2 were able to elicitan antibody response, with each of the three serotypes underinvestigation being represented by at least one immunogenic peptide. Thetiters of the immunogenic peptides were as follows:

TABLE 3 Titers of the immunogenic peptides in immunized mice. PeptideTiter M1-3 1:16,000  M3-1 1:8,000  M3-2 1:160,000 M12-1 1:160,000 M12-21:1000  

Mice are immunized s.c. with peptides and various combinations ofpeptides with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85,SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114,SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ IDNO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQID NO:138.

ELISA is used to confirm antibody elicitation to at least some of thepeptides and peptide combinations. Titers are determined for thepeptides and combinations of peptides.

Example 3 Opsonophagocytosis

The mouse sera that were shown to contain anti-M antibodies by the ELISAassay were then tested for functional activity with the in vitroopsonophagocytosis assay (Lancefield 1959), Table 4. Each of the seratested positive in the assay and was able to reduce the amount of viablebacteria relative to controls. The values for the reduction of bacteriain the opsonophagocytosis assays were as shown in Table 5.

TABLE 4 Sera results for opsonophagocytosis assay. Peptide Opsono (invitro) M1-4 nd M1-2 nd M1-3 yes M3-1 yes M3-2 yes M3-4 nd M12-1 yesM12-2 nd M12-3 nd

TABLE 5 Percentage reduction of viable GAS bacteria. Peptide ReductionM1-3 50% M3-1 80-90% M3-2 60-80% M12-1 70-80%It is interesting to note that within the M3 serotype, while the M3-1peptide induced an antibody response that was more than a magnitudelower than that of the M3-2 peptide, it was able to opsonophagocytizebacteria more effectively.

Mouse sera is tested that is shown to contain anti-M antibodies from thepeptides and combinations of peptides selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ IDNO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, SEQ ID NO:138 for functional activity with anin vitro opsonophagocytosis assay. The sera test positive in the assayand are able to reduce the amount of viable bacteria relative tocontrols.

Example 4 Nasopharyngeal Colonization Challenge

Nasopharyngeal (NP) colonization challenge experiments were performed onmice vaccinated (s.c.) with either M3-1 or M3-2 peptide in alum incomparison to control animals which received alum only. Thenasopharyngeal colonization challenge was performed by doing a nasalwash 24 hours after challenge. The subjects were administered 10⁴ CFUs 1week after final boost. Dilutions of the wash were plated. Both peptideswere able to induce an effective in vivo immune response that reducedcolonization in the vaccinated group relative to the unvaccinated group.

TABLE 6 Reduction of nasopharyngeal colonization in vaccinated micerelative to control mice. Reduction of Peptide colonization P M3-1 87% P= 0.010 M3-2 67% P = 0.029The M3-1 peptide reduced NP colonization by 87% (P<0.010), while theM3-2 peptide was able to reduce NP colonization by 67% (P<0.029)relative to the control group. No deaths were recorded in any group. Toour knowledge this is the first example of in vivo reduction ofnasopharyngeal colonization of GAS bacteria by immunization with atype-specific synthetic peptide.

NP colonization challenges are performed on mice vaccinated s.c. withthe peptides and peptide combinations selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ IDNO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, and SEQ ID NO:138. Peptides and peptidecombinations are confirmed to induce an effective in vivo immuneresponse that reduced colonization in the vaccinated group relative tothe unvaccinated group. No deaths are reported in any group.

Example 5 Cross-Type Protection by Peptides

Some of the peptides tested in the previous examples were tested forcross-type protection using the methods described above. Table 7 showsin vitro data on reduction in bacteria versus a control. The peptidetested and serotype against which it was tested is indicated. Thisexample clearly shows that peptides M3-1 and M3-2 were able tosignificantly reduce M43 type bacteria in addition to M3 type bacteria.

TABLE 7 Functional antibody bactericidal activity in vitro showingcross-protection against heterologous M type. GAS M type used % CFUPeptide in assay reduction M1-3 M1 80-95% M3-1 M3 70-90% M3-2 M3 90-95%M12-1 M12 70-80% M3-1 M43 80-90% M3-2 M43 60-80%

Some of the peptides were tested for reduction of colonization of GAS inmice. The second line of data shows mice immunized with peptides M1-3,M3-2 and M12-1. The peptides were administered together but were notchemically combined in any way. The third line of data shows mice thatwere inoculated with M3-2. Some of the mice were colonized by M3, andsome of the mice were colonized by M43. The % CFU reduction is thereduction overall for the mice which received M3-2. This againdemonstrates that M3-2 was capable in vivo of cross-type protection.

TABLE 8 Reduction of colonization in mice. Colonizing GAS % CFUreduction Peptides M type (P value) M1-3 M1 90% (P < 0.10) M1-3, M1 74%(P < 0.25) M3-2, M12-1 M3-2 M3 or M43 67% (P < 0.30)

The cross-type protection is confirmed for peptides and peptidecombinations selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59,SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79,SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99,SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113,SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ IDNO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127,SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ IDNO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQID NO:137, and SEQ ID NO:138. Reductions in bacteria are shown in vitroand in vivo.

Example 6 Detection of Heart Cross-Reactive Antibodies

Mouse immune sera that contained functional antibody were examined forthe presence of heart cross-reactive antibodies with an IFA. The mousesera were tested in parallel with a positive control, mouse monoclonalantibody to human leukocyte antigen (HLA). None of the anti-M peptidesera from immunized mice reacted with the heart tissue.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

We claim:
 1. A composition comprising a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 32. 2. The composition of claim 1, whereinthe polypeptide is a chimeric fusion protein.
 3. The composition ofclaim 2, wherein the chimeric fusion protein comprises an amino acidsequence of one or more heterologous Group A Streptococcus (GAS) M-typepeptides.
 4. The composition of claim 1, wherein the polypeptide islinked to a peptide backbone.
 5. The composition of claim 1, furthercomprising a pharmaceutically acceptable vehicle or carrier.
 6. Thecomposition of claim 1, further comprising additional immune-stimulatorymolecules.
 7. The composition of claim 6, wherein the additionalimmune-stimulatory molecules comprise GAS-based peptides, non-GASimmunogens selected from the group consisting of H. influenza,pertussis, N. meningitidis, pneumococcus, and Influenzae, or adjuvants.8. The composition of claim 1, wherein the polypeptide consistsessentially of the amino acid sequence of SEQ ID NO:
 32. 9. Thecomposition of claim 1, wherein the polypeptide consists of the aminoacid sequence of SEQ ID NO:
 32. 10. An immunogenic composition foreliciting an immune response to Group A Streptococcus comprising animmunogenic amount of the composition of claim
 1. 11. The immunogeniccomposition of claim 10, wherein the immunogenic composition is capableof eliciting functional opsonic antibodies without eliciting tissuecross reactive antibodies.
 12. The immunogenic composition of claim 10,wherein the immunogenic composition is effective in decreasing thenasopharyngeal reservoir of GAS when administered.
 13. A method forinducing an immune response against the emm12 serotype of Group AStreptococcus, comprising administering an immunogenic amount of theimmunogenic composition of claim 1 to a subject.
 14. The method of claim13, wherein the administration is via injection or a mucosal deliverymethod.
 15. The method of claim 13, wherein the immune responsecomprises induction of opsonic antibodies.
 16. An isolated polypeptidecomprising the amino acid sequence according to SEQ ID NO:
 32. 17. Thepolypeptide of claim 16, wherein the polypeptide is a chimeric fusionprotein.
 18. The polypeptide of claim 17, wherein the chimeric fusionprotein comprises an amino acid sequence of one or more heterologousGroup A Streptococcus (GAS) M-type peptides.
 19. The isolatedpolypeptide of claim 16, wherein the polypeptide consists essentially ofthe amino acid sequence of SEQ ID NO:
 32. 20. The isolated polypeptideof claim 16, wherein the polypeptide consists of the amino acid sequenceof SEQ ID NO: 32.